Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3ß, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Differential Ubiquitination as an Effective Strategy Employed by the Blood-Brain Barrier for Prevention of Bacterial Transcytosis.

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of pathogens into central nervous system (CNS) are critical for maintenance of brain homeostasis. These include various intracellular defense mechanisms that are vital to block transcytosis of neurotropic pathogens into the CNS. However, mechanistic details of coordination between these defense pathways remain unexplored. In this study, we established that BBB-driven ubiquitination acts as a major intracellular defense mechanism for clearance of Streptococcus pneumoniae, a critical neurotropic pathogen, during transit through BBB. Our findings suggest that the BBB employs differential ubiquitination with either K48- or K63-ubiquitin (Ub) chain topologies as an effective strategy to target S. pneumoniae toward diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs S. pneumoniae exclusively toward proteasomes. Time-lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in the BBB, the majority of the ubiquitinated S. pneumoniae was cleared by proteasome. Fittingly, inhibition of proteasome and autophagy pathway led to accumulation of K48-Ub- and K63-Ub-marked S. pneumoniae, respectively, and triggered significant increases in intracellular S. pneumoniae burden. Moreover, genetic impairment of either K48- or K63-Ub chain formation demonstrated that although both chain types are key in disposal of intracellular S. pneumoniae, K48-Ub chains and subsequent proteasomal degradation have more pronounced contributions to intracellular S. pneumoniae killing in the BBB. Collectively, these observations, for the first time, illustrated a pivotal role of differential ubiquitination deployed by BBB in orchestrating a symphony of intracellular defense mechanisms for interception and degradation of S. pneumoniae, blocking its entry into the brain, which could be exploited to prevent bacterial CNS infections. IMPORTANCE The blood-brain barrier (BBB) represents a unique cellular barrier that provides structural integrity and protection to the CNS from pathogen invasion. Recently, ubiquitination, which is key for cellular homeostasis, was shown to be involved in pathogen clearance. In this study, we deciphered that the BBB deploys differential ubiquitination as an effective strategy to prevent S. pneumoniae trafficking into the brain. The different ubiquitin chain topologies formed on S. pneumoniae dictated the selection of downstream degradative pathways, namely, autophagy and proteasomes, among which the contribution of the proteasomal system in S. pneumoniae killing is more pronounced. Overall our study revealed how the BBB deploys differential ubiquitination as a strategy for synchronization of various intracellular defense pathways, which work in tandem to ensure the brain's identity as an immunologically privileged site.